Mitochondria are centers of metabolism and signaling whose function is essential to all but a few eukaryotic cell types. General dysfunction of these organelles is implicated in a wide range of inborn errors of metabolism, and in an increasing number of common human diseases, including type 2 diabetes (T2D), cancer and heart disease. However, the specific alterations that underlie mitochondrial dysfunction in these disorders are most often poorly defined, and are nearly always impervious to therapeutic intervention. As such, clearly defining the pathogenic mitochondrial alterations that underlie metabolic disorders and devising new therapeutic strategies to treat these conditions represent principal challenges in mitochondrial medicine. Emerging evidence, including our own recent proteomic data, have revealed that mitochondrial proteins are replete with phosphorylation and acetylation sites that change dynamically between healthy and diseased states, and that these modifications are frequently found jointly on the same proteins. These data suggest that these post- translational modifications (PTMs) are widely important in regulating mitochondrial metabolism, and that aberrant levels of these modifications are among the relevant alterations underlying mitochondrial pathophysiology. If true, this could motivate the development of a novel therapeutic strategy: the control of mitochondrial metabolism via manipulation of cellular signaling processes. However, despite these promising early studies on mitochondrial PTMs, our understanding of their role in mitochondrial physiology remains in its infancy for a number of reasons. First, it remains unclear which of these modifications are actually important for regulating protein function. Second, there is a striking lack of information regarding the enzymes (e.g., kinases) that perform these modifications. These substantial knowledge gaps prevent us from understanding how cells use PTMs to manipulate mitochondrial function, and from exploiting this information for potential therapeutic benefit. This proposal takes a thorough and innovative approach to addressing these knowledge gaps by blending focused biochemistry with a range of state-of-the-art mass spectrometry tools. In particular, the overarching goals of this proposal are to 1) elucidate how phosphorylation affects the activities of select liver mitochondrial proteins involved in ketogenesis and beta-oxidation using in vitro biochemistry and cell-based metabolomics and bioenergetics, 2) to identify kinases in mitochondria that execute these events through activity-based and targeted mass spectrometry techniques, and 3) to establish a complementary, quantitative map of the dynamic mitochondrial protein acetylation events that accompany the onset of obesity and T2D. Completion of these aims will provide new insight into the regulation of key mitochondrial metabolic pathways, will provide a foundation for future mechanistic studies into the interrelationship between mitochondrial post-translational modifications, and will help reveal new therapeutic targets for the treatment of mitochondrial dysfunction.